Superoxide is a major reactive oxygen species (ROS) in the biological system and can be converted to other ROS such as hydrogen peroxide catalyzed by superoxide dismutase (SOD). Our studies during the previous grant period demonstrated that inhibition of SOD preferentially kills cancer cells by ROS-mediated mechanism, and that this selective anticancer activity seems attributable to the intrinsic ROS stress in cancer cells. We have also identified two natural compounds that cause striking increase of ROS generation in Ras oncogenic transformed cells, leading to preferential killing of these malignant cells. These research findings, together with studies by other groups, provide a compelling rationale for developing novel therapeutic strategies to selectively kill cancer cells based on their intrinsic oxidative stress. However, the fundamental questions why cancer cells are under intrinsic oxidative stress and how these novel compounds selectively kill the malignant cells remain unanswered. This research proposal is designed to address several key issues in this area. We will use novel experimental systems to test the hypothesis that oncogenic signals and mitochondrial malfunction play key roles in ROS generation and in promoting malignant cellular behaviors, and that these abnormalities can be specifically targeted by novel pharmacological agents, leading to selective killing of the malignant cells by ROS-mediated mechanisms. The specific aims of this research are: (1) Examine the role of mitochondria in mediating Ras-induced ROS generation and in amplification of ROS stress in cancer cells. A novel dominant-negative mitochondrial pol gamma (POLGdn) expression system will be used as a unique tool to evaluate the contribution of mitochondrial respiration chain in mediating oncogene-induced ROS generation. (2) Examine the novel mechanism by which ROS promote cancer cell motility and invasion through a redox-sensitive upregulation of CXCL14 expression, leading to elevated cell migration (3) Test the RAS oncogenic pathway and related molecules as novel therapeutic targets for selective killing of cancer cells through ROS-mediated mechanisms. (4) Evaluate the potential therapeutic activity of two novel natural compounds against pancreatic cancer or ovarian cancer cells with constitutive Ras oncogenic activation. We anticipate that this research will provide significant new insights into the mechanisms responsible for increased oxidative stress in cancer cells, especially the fundamental roles of oncogenic signals and mitochondria in regulating ROS generation, promoting malignant cell behaviors, and affecting cancer cell response to therapeutic agents. The investigation of novel compounds for their ability to selectively kill oncogenic transformed cancer cells through ROS-mediated mechanisms will potentially have significant therapeutic implications, owing to their promising activity against pancreatic cancer and ovarian cancer, and their low toxicity in normal cells. [unreadable] [unreadable] [unreadable] [unreadable]